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Copper transport by lobster (Homarus americanus) hepatopancreatic mitochondria

Pamela Chavez-Crooker^*, Nestor Garrido and Gregory A. Ahearn

Department of Zoology, 2538 The Mall, University of Hawaii at Manoa, Honolulu, HI 96822, USA
^* Present address: Laboratory of Biotechnology and Molecular Biology, Facultad Recursos del Mar, Universidad de Antofagasta, Casilla 170, Antofagasta, Chile
Present address: Laboratory of Biological Chemistry, Department of Chemistry, Universidad Catolica del Norte, Casilla 1280, Antofagasta, Chile

View larger version (17K): [in a new window]   Fig. 1. Time course of Phen Green (1 µmol l–1) fluorescence quenching induced by addition of copper chloride at three different concentrations to buffer containing dye-equilibrated hepatopancreatic mitochondria. Arrows show the points where copper chloride was added to a stable fluorescence signal produced in the absence of the metal. This graph is representative of multiple similar recordings under experimental conditions reported in this study. Copper influx was estimated by the initial rate of fluorescence quenching observed over the first 120 s of incubation following addition of the metal. The excitation wavelength was 490 nm and the emission of the dye was recorded at 520 nm. Addition of 1 mmol l–1 EDTA restored the fluorescent signal by complexing Cu2+ in solution.

View larger version (17K): [in a new window]   Fig. 2. Effect of 500 µmol l–1 calcium chloride on the kinetics of Cu2+ influx (measured as F mg–1 protein 120 s–1, where F is fluorescence quenching) in purified lobster hepatopancreatic mitochondrial suspensions. Concentrations of Cu2+ used in the experiment were 0.4, 1.6, 4.1, 8.2 and 12.2 µmol l–1. Previous studies have shown that Ca2+ alone has no effect on the quenching phenomenon (Chavez-Crooker et al., 2001). Best-fit lines were drawn using SigmaPlot software, and the resulting kinetic constants are presented in the text. Individual triplicate values are displayed on the figure.

View larger version (18K): [in a new window]   Fig. 3. Effect of Ruthenium Red on 1 µmol l–1 Phen Green fluorescence quenching. The activated form (which does not have to enter mitochondria and be activated by enzymes) of the dye was used in buffer containing the following concentrations of Ruthenium Red (RR): 0, 250, 500, 1000, 2500 and 5000 nmol l–1. Fluorescence quenching (F) was followed over 120 s after the addition of Ruthenium Red. Changes in initial fluorescence are presented in figure. Values are means ± S.E.M., N=3. From this figure, only 5 % of the initial signal was lost at a Ruthenium Red concentration of 500 nmol l–1, but 50 % was abolished when the inhibitor concentration was raised to 5000 nmol l–1. The regression equation is F=543.6–0.057[RR], r2=0.99, P=0.001.

View larger version (18K): [in a new window]   Fig. 4. Effect of 500 nmol l–1 Ruthenium Red (RR) on the kinetics of Cu2+ influx into purified mitochondrial suspensions from lobster hepatopancreas. Data are presented as in Fig. 2. Best-fit lines were drawn using SigmaPlot software, and the resulting kinetic constants are presented in the text. Individual triplicate values are displayed on the figure.

View larger version (18K): [in a new window]   Fig. 5. Effect of 500 µmol l–1 diltiazem on the kinetics of Cu2+ influx into purified mitochondrial suspensions from lobster hepatopancreas. Data are presented as in Fig. 2. Best-fit lines were drawn using SigmaPlot software, and the resulting kinetic constants are presented in the text. Individual triplicate values are displayed on the figure.

View larger version (38K): [in a new window]   Fig. 6. Working model of Cu2+ uptake by lobster hepatopancreatic epithelial cells and sequestration within hepatopancreatic mitochondria by transport mechanisms described in the present investigation and reported previously (Klein and Ahearn, 1999; Chavez-Crooker et al., 2001). Cu2+ has previously been shown to be transported across epithelial brush-border membranes (BBMs) by an antiport process that exchanges external Cu2+ with intracellular cations such as Na+ on an electroneutral, amiloride-insensitive 1Cu2+/2Na+ antiporter. Ca2+ derived from the luminal medium may enter the cells through a verapamil-sensitive channel and either allosterically activate the exchanger or serve as a transport substrate. Intracellular Ca2+ and Cu2+ may enter hepatopancreatic mitochondria via three possible pathways: (i) an electrogenic, Ruthenium-Red-sensitive uniporter, (ii) a diltiazem-sensitive, electroneutral 1Ca2+(1Cu2+)/2Na+ exchanger and a diltiazem-insensitive, electroneutral 1Ca2+(1Cu2+)/2H+ exchanger. Ruthenium Red (RR) appears to interact with all three transport processes. Dilt, diltiazem.